1. Field of the Invention
The present invention relates generally to the fields of biology and medicine. More particularly, the present invention is directed to compounds, methods and compositions useful in increasing in mammals the transport and delivery of hydrophilic molecules having an amino group, in particular peptides and proteins.
2. Related Art
Advances in biochemistry have made possible the production of large amounts of therapeutically active and pure proteins and peptides. Currently, the therapeutic effects of most of these agents can be achieved only when they are administered via invasive routes, such as by injection. Since most proteins have very short half lives, effective concentrations of these agents can be maintained only when administered by frequent injections.
Although the administration of protein by injection is the most effective means of their delivery in vivo, patient tolerance of multiple injections is very poor. In addition, drug injection requires training and skill that may not always be transferable to patients. In cases where protein drugs have a life-saving role, the administration by injection can be acceptable by the patients. However, in cases where protein drugs are just one of several possible therapies, injections of proteins and peptides are unlikely to be accepted by the patients. Therefore, alternative routes of protein and peptide delivery need to be developed.
Such alternative routes may include the buccal, nasal, oral, pulmonary, rectal and ocular routes. Without exception, these routes are less effective than the parenteral routes of administration, but are still far more attractive than the parenteral routes because they offer convenience and control to the patients. The oral route is particularly attractive because it is the most convenient and patient-compliant.
Mucosal barriers, which separate the inside of the body from the outside (e.g., gastrointestinal, ocular, pulmonary, rectal and nasal mucosa), comprise a layer of tightly joined cell monolayers which strictly regulate the transport of molecules. Individual cells in barriers are joined by tight junctions which regulate entry into the intercellular space. Hence, the mucosa is at the first level a physical barrier, transport through which depends on either the transcellular or the paracellular pathways (Lee, V. H. L., Critical Rev. Ther. Drug Delivery Sys. 5:69-97 (1988)).
Paracellular transport through water filled tight junctions is restricted to small molecules (MW less than 1 kDa) and is essentially a diffusion process driven by a concentration gradient across the mucosa (Lee, V. H. L., Critical Rev. Ther. Drug Delivery Sys. 5:69-97 (1988); Artursson, P. and Magnusson, C., J. Pharm. Sci. 79:595-600 (1990)). The tight junctions comprise less than 0.5% of the total surface area of the mucosa (Gonzalez-Mariscal, L. M., et al., J. Membrane Biol. 86:113-125 (1985); Vetvicka, V. and Lubor, F., Critical Rev. Ther. Drug Deliv. Sys. 5:141-170 (1988)); therefore, they play only a minor role in the transport of protein drugs across the mucosa.
The transcellular transport of small drugs occurs efficiently provided the physicochemical properties of the drug are suited to transport across hydrophobic cell barriers. However, the transcellular transport of proteins and peptides is restricted to the process of transcytosis (Shen, W. C., et al., Adv. Drug. Deliv. Rev. 8:93-113 (1992)). Transcytosis is a complex process in which proteins and peptides are taken up into vesicles from one side of a cell, and are subsequently shuttled through the cell to the other side of the cell, where they are discharged from the endocytic vesicles (Mostov, K. E. and Semister, N. E., Cell 43:389-390 (1985)). The cell membrane of mucosa barriers is a hydrophobic lipid bilayer which has no affinity for hydrophilic, charged macromolecules like proteins and peptides. In addition, mucosa cells may secrete mucin which can act as a barrier to the transport of many macromolecules (Edwards, P., British Med. Bull. 34:55-56 (1978)). Therefore, unless specific transport mechanisms exist for proteins and peptides, their inherent transport across mucosa barriers is almost negligible.
In addition to providing a tight physical barrier to the transport of proteins and peptides, mucosa barriers possesses enzymes which can degrade proteins and peptides before, after, and during their passage across the mucosa. This barrier is referred to as the enzymatic barrier. The enzymatic barrier consists of endo- and exopeptidase enzymes which cleave proteins and peptides at their terminals or within their structure. Enzymatic activity of several mucosa have been studied and the results demonstrated that substantial protease activity exists in the homogenate of buccal, nasal, rectal and vaginal mucosa of albino rabbits and that these activities are comparable to those present in the ilium (Lee, V. H. L., Critical Rev. Ther. Drug Delivery Sys. 5:69-97 (1988)). Therefore, regardless of the mucosa being considered, the enzymatic barrier present will feature strongly in the degradation of the protein and peptide molecules.
The N and the C termini of peptides are charged and the presence of charged side chains imparts highly hydrophilic characteristics on these macromolecules. In addition, the presence of charged side chains means that proteins and peptides have strong hydrogen bonding capacities; this H-bonding capacity has been demonstrated to play a major role in inhibiting the transport of even small peptides across cell membranes (Conradi, R. A., et al., Pharm. Res. 8:1453-1460 (1991)). Therefore, the size and the hydrophilic nature of proteins and peptides combine to severely restrict their transport across mucosa barriers.
One approach that has been used to alter the physical nature of the mucosa barriers is the use of penetration enhancers. The use of penetration enhancers is based on the disruption of the cell barriers by low molecular weight agents which can fluidize cell membranes (Kaji, H., et al., Life Sci. 37:523-530 (1985)), open tight junctions (Inagaki, M., et al., Rhinology 23:213-221 (1985)), and create pores in the cell membrane (Gordon, S., et al., Proc. Natl. Acad. Sci. USA 82:7419-7423 (1985); Lee, V. H. L., et al., Crit. Rev. Ther. Drug. Carrier Syst. 8:91-192 (1991)). The use of these agents leads to a non-specific loss of barrier integrity and can lead to the absorption of a variety of large molecules which can be toxic to cells in vivo.
Protease inhibitors have been co-administered with proteins and peptides and have shown some limited activity in enhancing the absorption of these macromolecules in vivo (Kidron, M., et al., Life Sci. 31:2837-2841 (1982); Takaroi, K., et al., Biochem. Biophys. Res. Comm. 137:682-687 (1986)). The safety and the long-term effects of this approach have yet to be thoroughly investigated.
The prodrug approach is based on the modification of peptides in a manner that will protect them from enzyme degradation and recognition. This has been achieved by the blockage of vulnerable groups on peptides by amidation and acylation. The prodrug approach has thus far proven useful only for small peptides which have easily identifiable domains of activity.
Reduction in size is another feasible approach to increasing the transport potential of proteins. However, the active sites of proteins need to be mapped before size reduction can be attempted. In general, this approach is difficult to apply to the majority of proteins.
Carrier ligands, by virtue of their properties, can alter the cell uptake and transport characteristics of proteins and peptides. The essence of this approach is that a cell-impermeant protein or peptide is covalently attached to a carrier which is highly transported into cells. The mechanisms through which carrier ligands became endocytosed and transcytosed are important in deciding the suitability of the carrier for enhancing the transport of proteins and peptides. Macromolecular carriers are hydrophilic and do not partition into the membrane. Therefore, the transport of large polymeric carriers into the cells is mediated by the affinity of the carrier for the cell membrane. Generally, the uptake of a macromolecular conjugate starts with binding to the cell membrane. The binding of the carrier to the cells can be specific (e.g., binding of antibodies to cell surface antigens), nonspecific (binding of cationic ligand or lectins to cell surface sugars), or receptor mediated (binding of transferring or insulin to their receptors). Once the carrier is bound to the cell surface, it is taken up into vesicles. These vesicles then become processed stepwise and can be routed to several pathways. One pathway is the recycling of the vesicle back to the membrane. Another pathway, which is destructive to the conjugate, is the fusion with lysosomes. An alternative pathway, and one which leads to the transcytosis of the conjugate, is the fusion of the vesicle with the membrane opposite to the side from which it was derived.
The correct balance between the processes of endocytosis and transcytosis determine the delivery of a protein conjugate to its target. For instance, endocytosis may determine the extent to which a conjugate is taken up by the target cell, but transcytosis determines whether or not a conjugate reaches its target (Shen, W. C., et al., Adv. Drug. Deliv. Rev. 8:93-113 (1992)). For successful absorption through the gastrointestinal tract, aconjugate must bind the apical membrane of the gastrointestinal mucosa, become internalized into the mucosa cells, be delivered across the cells, and finally become released from the basolateral membrane.
The current literature contains many reports which demonstrate that nonspecific carriers, such as polylysines (Shen, W. C. and Ryser, H. J. P., Proc. Natl. Acad. Sci. USA 78:7589-7593 (1981)) and lectins (Broadwell, R. D., et al., Proc. Natl. Acad. Sci. USA 85:632-646 (1988)), and specific carriers, such as transferrin (Wan, J., et al., J. Biol. Chem. 257:13446-13450 (1992)), asialoglycoprotein (Seth, R., et al., J. Infect. Diseases 168:994-999 (1993)), and antibodies (Vitetta, E. S., J. Clin. Immunol. 10:15S-18S (1990)) can enhance the endocytosis of proteins into cells. Reports dealing with transcytotic carriers for proteins are fewer, and very few studies have quantitated the transport of protein conjugates across cell barriers. Wheat germ agglutinin (Broadwell, R. D., et al., Proc. Natl. Acad. Sci. USA 85:632-646 (1988)) and an anti-transferrin/methotrexate conjugate (Friden, P. M. and Walus, L. R., Adv. Exp. Med. Biol. 331:129-136 (1993)) have been shown to be transcytosed across the blood-brain barrier in vivo. Also, polylysine conjugates of horseradish peroxidase (HRP) and a transferrin conjugate of HRP have been shown to be transcytosed across cell monolayers in vitro (Wan, J. and Shen, W. C., Pharm. Res. 8:S-5 (1991); Taub, M. E. and Shen, W. C., J. Cell. Physiol. 150:283-290 (1992); Wan, J., et al., Biol. Chem 267:13446-13450 (1992)).
Fatty acids, as constituents of phospholipids, make up the bulk of cell membranes. They are available commercially and are relatively cheap. Due to their lipidic nature, fatty acids can easily partition into and interact with the cell membrane in a non-toxic way. Therefore, fatty acids represent potentially the most useful carrier ligand for the delivery of proteins and peptides. Strategies that may use fatty acids in the delivery of proteins and peptides include the covalent modification of proteins and peptides and the use of fatty acid emulsions.
Some studies have reported the successful use of fatty acid emulsions to deliver peptide and proteins in vivo (Yoshikawa, H., et al., Pharm. Res. 2:249-251 (1985); Fix, J. A., et al., Am. J. Physiol. 251:G332-G340 (1986)). The mechanism through which fatty acid emulsions influence the absorption of proteins and peptides is not yet known. Fatty acid emulsions may open tight junctions, solubilize membranes, disguise the proteins and peptides from the gastrointestinal environment, and carry proteins and peptides across the gastrointestinal mucosa as part of their absorption (Smith, P., et al., Adv. Drug. Delivery Rev. 8:253-290 (1992)). The latter mechanism has been proposed, but is inconsistent with current knowledge about the mechanism of fat absorption.
A more logical strategy to deliver proteins and peptides across the gastrointestinal epithelium is to make use of fatty acids as non-specific membrane adsorbing agents. Several studies have shown that a non-specific membrane binding agent linked to a protein can promote the transcytosis of a protein conjugate across cells in vitro (Wan, J., et al., J. Cell. Physiol. 145:9-15 (1990); Taub, M. E. and Shen, W. C., J. Cell. Physiol. 150:283-290 (1992)). Fatty acid conjugation has also been demonstrated to improve the uptake of macromolecules into and across cell membranes (Letsinger, R., et al., Proc. Natl. Acad. Sci. USA 86:6553-6556 (1989); Kabanov, A., et al., Protein Eng. 3:39-42 (1989)). Nonetheless, there have been difficulties in conjugating fatty acids to peptides and proteins, including: (1) the lack of solubility of fatty acids in the aqueous solution for the conjugation reaction; (2) the loss of biological activity of peptides and proteins after fatty acid acylation; and (3) the lack of solubility of fatty acid-conjugated peptides in aqueous solutions (see, e.g., Hashimoto, M., et al., Pharm. Res. 6:171-176 (1989); Martins, M. B. F., et al., Biochimie 72:671-675 (1990); Muranishi, S., et al., Pharm. Res. 6:171-176 (1989); Martins, M. B. F., et al., Biochimie 72:671-675 (1990); Muranishi, S., et al., Pharm. Res. 8:649-652 (1991); Robert, S., et al., Biochem. Biophys. Res. Commun. 196:447-454 (1993)).
Once delivered into the cell, peptides and proteins must be released from their carrier. Published PCT Application Nos. WO 96/22773 and WO 98/13007 disclose the transcellular delivery and release of sulfhydryl-containing peptides and proteins. The cellular absorption of sulfhydryl-containing hydrophilic molecules can be increased by conjugation with a fatty acid through a disulfide linkage. The labile disulfide linkage is easily reduced, providing a mechanism for the release of the hydrophilic compounds from the fatty acid moiety once inside the body.
In addition to disulfide bond reduction, other mechanisms for the release of biologically active hydrophilic compounds from carrier systems include hydrolysis and photolytic bond cleavage. (See for example, U.S. Pat. No. 5,505,931 and references cited therein). Hydrolysis-based delivery systems in which a biologically active amine is conjugated with an organic acid incorporating a monoclonal antibody or other substrate for the targeting of specific cells are known. (See U.S. Pat. Nos. 4,764,368, 4,618,492, 5,505,931 and 5,563,250). After specific binding to the targeted cell, these conjugates deliver the active amine (typically in the form of an amide) inside the cell where hydrolysis (of the amide) releases the free amine inside the cell.
The success of prior art hydrolysis-based delivery systems has inspired the search for improved drug-carrier conjugates capable of delivering a biologically active amino group containing compound to the inside of cells. Improved synthetic strategies and treatment techniques are currently being developed.
The present invention relates to new drug-carrier conjugates and convenient synthetic strategies for their production. Accordingly, the present invention is directed to synthetic methods, intermediates and ultimately final products useful for the uptake and release of biologically-active amino group containing compounds.
In particular, the invention relates to compounds of general Formula I 
in which R2 is selected from the group consisting of hydrogen, halo, alkyl, or aryl, wherein the alkyl or aryl groups are optionally substituted with one or more alkoxy, alkoxyalkyl, alkanoyl, nitro, cycloalkyl, alkenyl, alkynyl, alkanoyloxy, alkyl or halogen atoms;
R3 is a lipophilic group;
one of R4 and R5 is a biologically active amino group containing substance selected from the group consisting of an amine-containing drug, a natural or unnatural amino acid, a peptide and a protein and the other of R4 and R5 is OR6 where R6 is hydrogen, an alkali metal or a negative charge;
X is oxygen or sulfur;
Y is a bridging natural or unnatural amino acid;
n is zero or 1; and
m is an integer from zero to 10.
The present invention also relates to compounds of the general Formula II 
in which R2 is hydrogen, halo, alkyl, or aryl, wherein the alkyl and aryl groups are optionally substituted with one or more alkoxy, alkoxyalkyl, alkanoyl, nitro, cycloalkyl, alkenyl, alkynyl, alkanoyloxy, alkyl or halogen atoms;
R3 is a lipophilic group;
X is O or S;
Y is a bridging natural or unnatural amino acid;
n is zero or 1; and
m is an integer from zero to 10.
The present invention also relates to compounds of the general Formula III 
or a pharmaceutically-acceptable salt thereof, in which R2 is hydrogen, halo, alkyl, or aryl, wherein the alkyl and aryl groups are optionally substituted with one or more alkoxy, alkoxyalkyl, alkanoyl , nitro, cycloalkyl, alkenyl , alkynyl, alkanoyloxy, alkyl or halogen atoms;
R3 is a lipophilic group;
X is O or S;
Y is a bridging natural or unnatural amino acid;
n is zero or 1; and
m is an integer from zero to 10.
The present invention also relates to methods of forming conjugates of general Formula I from compounds of general Formula II and a biologically active amino group containing substance.
The present invention also relates to methods of forming compounds of general Formula II from maleic acid derivatives and the corresponding thiols or alcohols.
The present invention also relates to methods for increasing the absorption or prolonging blood and tissue retention in a mammal of a biologically active amino group containing substance, in which a conjugate of general Formula I is administered to the mammal in a pharmaceutically-acceptable form.
The present invention also relates to methods for increasing the delivery of hydrophilic amine containing compounds to the inside of a cell having a mucosal barrier, in which a conjugate of general Formula I is contacted with the cell whereby the conjugate penetrates the mucosal barrier of the cell and the free amine is liberated by hydrolysis of an amide bond.
The present invention also relates to pharmaceutical compositions comprising a compound of general Formula I.
The above and other features, advantages, embodiments, aspects and objects of the present invention will be clear to those skilled in the areas of relevant art, based upon the description, teaching and guidance presented herein.